Cell separation device and cell separation method

ABSTRACT

Plural types of cells having different dielectrophoretic properties are separated using a simple structure. There is provided a cell separation device including: a flow path through which a cell suspension flows, the cell suspension containing plural types of cells which have different dielectrophoretic properties; electrodes disposed to face each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path; an electric field gradient forming portion which generates an electric field strength gradient between the electrodes; and a power supply applying an alternating voltage having a direct current component across the electrodes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell separation device and a cell separation method.

This application is based on Japanese Patent Application Nos. 2007-213922, and 2008-197043, the content of which is incorporated herein by reference.

2. Description of Related Art

Heretofore, techniques for separating particles using differences in their dielectrophoretic properties have been known (for example, see PCT International Publications Nos. WO1997/34689 Pamphlet, WO2001/5512 Pamphlet, WO2001/5513 Pamphlet, and WO2001/5514 Pamphlet).

According to a technique disclosed in PCT International Publication No. WO1997/34689 Pamphlet, a plurality of traps formed of flat electrodes is disposed in a flow path, beads covered with antibodies which particularly adsorb certain bacteria are supported on the electrodes, and an effluent to be analyzed is made to flow through the flow path, so that bacteria approaching the vicinities of the electrodes are collected by the beads using dielectrophoretic properties.

According to a technique disclosed in PCT International Publication No. WO2001/5512 Pamphlet, a flow path is formed in which a plurality of wave-shaped electrodes is arranged, and plural types of particles are made to flow in the electrode arrangement direction, so that the particles are separated using the difference in dielectrophoretic properties.

According to a technique disclosed in PCT International Publication No. WO2001/5513 Pamphlet, after particles are transferred by application of ultrasonic waves, an electric field is applied so as to separate the particles by means of dielectrophoresis.

According to a technique disclosed in PCT International Publication No. WO2001/5514 Pamphlet, particles are separated by applying an electric field having at least two wavelengths so that a plurality of dielectrophoretic forces acts on the particles.

According to the technique disclosed in PCT International Publication No. WO1997/34689 Pamphlet, bacteria are quantitatively measured by the steps of preparing an analytical chamber at a downstream side in a flow path, measuring dielectrophoretic properties of beads before the bacteria are collected, separating beads that collect certain bacteria from the electrodes, and sending the beads thus separated into an analytical chamber for measurement to determine the difference in properties from beads that collect no bacteria; hence the process of this technique is disadvantageously complicated.

In addition, according to the technique disclosed in PCT International Publication No. WO 2001/5512 Pamphlet, in order to separate the particles flowing in the electrode arrangement direction, the wave-shaped electrodes must be used; hence, the device is complicated and expensive, and in addition, inconveniently, the separation cannot be efficiently performed.

In addition, the technique disclosed in PCT International Publication No. WO 2001/5513 Pamphlet disadvantageously requires additional energy such as ultrasonic waves.

Furthermore, the technique disclosed in PCT International Publication No. WO 2001/5514 Pamphlet is disadvantageously complicated since an electric field having two wavelengths is used.

BRIEF SUMMARY OF THE INVENTION

The present invention has been conceived in consideration of the above-described situation, and an object of the present invention is to provide a cell separation device and a cell separation method that can efficiently separate plural types of cells having different dielectrophoretic properties using a simple structure.

In order to accomplish the above object, the present invention provides the following solutions.

According to a first aspect of the present invention, there is provided a cell separation device comprising: a flow path through which a cell suspension flows, the cell suspension containing plural types of cells which have different dielectrophoretic properties; electrodes disposed to face each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path; an electric field gradient forming portion which generates an electric field strength gradient between the electrodes; and a power supply applying an alternating voltage having a direct current component across the electrodes.

According to the first aspect of the present invention, when an alternating voltage having a direct current component is applied across the electrodes by operation of the power source, an electric field having an electric field strength gradient is formed between the electrode by the operation of the electric field gradient forming portion, and in addition, charges are unevenly distributed at one electrode side due to the direct current component included in the alternating voltage. That is, the plural types of cells contained in the cell suspension all receive an electrophoretic force caused by the uneven distribution of charges. On the other hand, cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases, and cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases. In addition, a dielectrophoretic force is not applied to cells having no dielectrophoretic properties, such as dead cells, but only the electrophoretic force is applied thereto.

Accordingly, dielectrophoretic forces having different directions and an electrophoretic force can be applied to cells having different dielectrophoretic properties, and by maintaining an appropriate balance therebetween, the cells having different dielectrophoretic properties can be effectively separated from each other.

That is, for example, when the direction in which a negative dielectrophoretic force acts is set opposite to the direction in which an electrophoretic force acts, a relatively small force obtained by counteraction between the dielectrophoretic force and the electrophoretic force can be applied to cells having negative dielectrophoretic properties, and on the other hand, a relatively large force formed of only the electrophoretic force can be applied to cells having no dielectrophoretic properties. Hence, cells having no dielectrophoretic properties are attracted by the electrophoretic force, and cells having negative dielectrophoretic properties are made to flow along the flow direction, so that the cell separation can be performed.

In the above first aspect of the present invention, the electric field gradient forming portion may be an insulating member which has at least one opening and which is disposed between the electrodes.

With the above structure, the electric lines of force formed between the electrodes can be squeezed by the insulating member so as to pass through the opening, and an electric field having an electric field strength gradient can be easily formed between the electrodes.

In addition, in the above first aspect of the present invention, the electrodes may be parallel plate electrodes which form an electric field having an approximately uniform electric field strength.

With the above structure, according to the combination between the electrodes having a simple structure and the insulating plate, an electric field having an electric field strength gradient can be more easily formed between the electrodes.

In addition, in the above first aspect of the present invention, the electric field gradient forming portion may be constructed by forming one of the electrodes smaller than that of the other electrode.

With the above structure, by changing the density of the electric lines of force from the large electrode to the small electrode, an electric field having an electric field strength gradient can be easily formed.

In addition, according to a second aspect of the present invention, there is provided a cell separation method comprising the steps of: making a cell suspension containing plural types of cells having different dielectrophoretic properties flow in a flow path; and applying an alternating voltage including a direct current component across electrodes which faces each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path to form an electric field having an electric field strength gradient between the electrodes.

According to the above second aspect of the present invention, when an alternating voltage including a direct current component is applied across the electrodes, an electric field having an electric field strength gradient is formed between the electrodes, and at the same time, charges are unevenly distributed at one electrode side due to the direct current component included in the alternating voltage. That is, all types of cells contained in the cell suspension receive an electrophoretic force caused by the uneven distribution of charges. On the other hand, cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases, and cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases. In addition, to cells having no dielectrophoretic properties, such as dead cells, a dielectrophoretic force is not applied, but only the electrophoretic force is applied.

Accordingly, dielectrophoretic forces having different directions and an electrophoretic force can be applied to cells having different dielectrophoretic properties, and by maintaining an appropriate balance therebetween, the cells having different dielectrophoretic properties can be effectively separated from each other.

The present invention provides an advantage in that plural types of cells having different dielectrophoretic properties can be effectively separated with a simple structure.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is an overall schematic structural view showing a cell separation device according to one embodiment of the present invention.

FIG. 2 is a view showing an alternating voltage waveform generated by a power source of the cell separation device shown in FIG. 1.

FIG. 3A is a view showing electric lines of force formed between electrodes of the cell separation device shown in FIG. 1 and forces applied to cells, the view showing the state before separation.

FIG. 3B is a view showing electric lines of force formed between the electrodes of the cell separation device shown in FIG. 1 and forces applied to the cells, the view showing the state after separation.

FIG. 4 is a view illustrating an operation for separating cells having different dielectrophoretic properties with the cell separation device shown in FIG. 1.

FIG. 5 is a partial enlarged vertical cross-sectional view showing a modification of the cell separation device shown in FIG. 1.

FIG. 6 is a graph showing the change in frequency of an alternating voltage generated by using the cell separation device shown in FIG. 5.

FIG. 7 is a graph showing frequency properties at a cell retention boundary.

FIG. 8 is a graph showing retention boundaries and retention areas of living cells and dead cells.

FIG. 9 is an overall schematic structural view showing a cell separation device according to one Example of the present invention.

FIG. 10 is a schematic view of a porous insulating plate of the cell separation device according to the above Example of the present invention.

FIG. 11 is a view showing retention rates of living cells and dead cells.

DETAILED DESCRIPTION OF THE INVENTION

A cell separation device 1 according to one embodiment of the present invention will be described with reference to FIGS. 1 to 4.

The cell separation device 1 according to this embodiment includes, as shown in FIGS. 1 to 4, a flow path 2 through which a cell-suspension liquid containing plural types of cells A and B, such as living cells A and dead cells B, flows in one direction; electrodes 3 a and 3 b disposed in the flow path 2; an insulating plate (insulating member: electric field gradient forming member) 4 disposed between the electrodes 3 a and 3 b; and a power source 5 applying a voltage across the electrodes 3 a and 3 b.

The flow path 2 is divided by the electrically insulating plate 4 into a first flow path 2 a and a second flow path 2 b, both of which extend in a flowing direction.

The electrodes 3 a and 3 b are, for example, parallel plate electrodes and are disposed on facing wall surfaces of the flow path 2 to face each other in the direction perpendicular to a flow direction L.

In addition, the insulating plate 4 is formed to have a flat plate shape, is disposed at a central position of a space formed between the electrodes 3 a and 3 b, and has at least one through-hole (opening) 4 a.

Hence, the first flow path 2 a and the second flow path 2 b communicate with each other via the through-hole 4 a formed in the insulating plate 4.

Accordingly, since electric lines of force C generated from one side to the other side between the electrodes 3 a and 3 b by operating the power source 5 are blocked by the insulating plate 4, the electric lines of force C pass only through the through-hole 4 a formed in the insulating plate 4, as shown in FIGS. 3A and 3B. That is, since the electric lines of force C are bundled by the through-hole 4 a, and the density thereof is increased thereby, in an electric field formed between the electrodes 3 a and 3 b, a gradient is formed such that the electric field strength is highest at the through-hole 4 a and gradually decreases along the directions toward the electrodes 3 a and 3 b located at the two sides.

The power source 5 is, as shown in FIG. 2, designed to apply a sine-wave alternating voltage having a direct current component offset in one direction (such as plus (+) direction) across the electrodes 3 a and 3 b.

A cell separation method using the cell separation device 1 according to this embodiment, having the structure as described above, will now be described.

In order to separate the plural types of cells A and B having different dielectrophoretic properties using the cell separation device 1 according to this embodiment, an alternating voltage is applied across the electrodes 3 a and 3 b by operating the power source 5 to form the electric lines of force C, as shown in FIGS. 3A and 3B, between the electrodes 3 a and 3 b.

In the state described above, a cell-suspension liquid containing the plural types of cells A and B having different dielectrophoretic properties is made to flow in the first flow path 2 a. In this case, for example, the cell-suspension liquid is made to flow through the first flow path 2 a between the minus (−) side electrode 3 a and the insulating plate 4, and a medium containing no cells A and B is made to flow through the second flow path 2 b between the plus (+) side electrode 3 b and the insulating plate 4. The flow velocities of the cell-suspension liquid and the medium are set sufficiently slow.

Once the cells A having negative dielectrophoretic properties reach an area between the electrodes 3 a and 3 b, due to an electric field having an electric field strength gradient formed between the electrodes 3 a and 3 b, the cells A receive a dielectrophoretic force f1 in the direction along which the electric field strength decreases, as shown in FIGS. 3A and 3B. In addition, since the alternating voltage supplied from the power source 5 includes the direct current component, the cells A and B each receive an electrophoretic force f2 in the direction opposite to that of the dielectrophoretic force f1 in accordance with the charges of the cells A and B, as shown in FIGS. 3A and 3B. Hence, when the dielectrophoretic force f1 and the electrophoretic force f2, which are applied to the cells A, are balanced, the cells A having negative dielectrophoretic properties move smoothly along the flow of the cell-suspension liquid flowing in the first flow path 2 a without being attracted by the electrodes 3 a and 3 b.

On the other hand, since the dielectrophoretic force f1 is not applied to the cells B having no dielectrophoretic properties, the electrophoretic force f2 is only applied to the cells B. As a result, due to the electrophoretic force f2, the cells B are attracted by the plus (+) side electrode 3 b and flow into the second flow path 2 b through the through-hole 4 a formed in the insulating plate 4.

That is, by operating the power source 5, the amplitude and frequency of the alternating voltage applied across the electrodes 3 a and 3 b are adjusted so as to adjust the dielectrophoretic force f1 applied to the cells A having dielectrophoretic properties, and the absolute value of the direct current component included in the alternating voltage is adjusted so as to adjust the electrophoretic force f2 applied to the cells A and B. Accordingly, when the sum of the dielectrophoretic force f1 and the electrophoretic force f2 applied to the cells A, and the electrophoretic force f2 applied to the cells B are appropriately adjusted, it is possible to form a flow of the cells A flowing in the first flow path 2 a without being interrupted and a flow of the cells B introduced into the second flow path 2 b by being attracted by the electrode 3 b, as shown in FIG. 4; hence, the two types of cells can be effectively separated.

As described above, according to the cell separation device 1 and the cell separation method of this embodiment, by adopting the structure in which only the parallel plate electrodes 3 a and 3 b and the insulating plate 4 are disposed in the flow path 2 through which the cell-suspension liquid flows, it is possible to obtain an advantage in that the plural types of cells A and B having different dielectrophoretic properties can be efficiently separated from each other.

In this embodiment, although at least one through-hole 4 a is provided in the insulating plate 4 which is disposed between the electrodes 3 a and 3 b, the number of the through-holes 4 a is not particularly limited. In addition, the shape, arrangement, and intervals of the through-holes 4 a may be arbitrarily determined as long as a sufficient electric field gradient can be formed.

In addition, although the insulating plate 4 is disposed at the central position of the space formed between the parallel plate electrodes 3 a and 3 b, instead of this arrangement, as shown in FIG. 5, the insulating plate 4 may be disposed in close contact with the surface of one electrode 3 a of the above parallel plate electrodes 3 a and 3 b. In this case, in the electrode 3 a covered with the insulating plate 4, the electric lines of force C are formed only from the portion exposed through the through-hole 4 a, and the electric field is formed such that the electric field strength gradually decreases in the direction toward the other electrode 3 b. Accordingly, as in the case described above, the cells A and B having different dielectrophoretic properties can be separated in the direction intersecting the flow direction L in the flow path 2.

Hereinafter, an Example using the cell separation device 1 shown in FIG. 5 will be described with reference to FIG. 6.

As the electrodes 3 a and 3 b, a pair of flat plate-shaped electrodes 3 a and 3 b made of titanium having a thickness of 1 mm was used. The space between the electrodes 3 a and 3 b was set to approximately 1 mm. In addition, as the insulating plate 4, a Kapton film having a thickness of 50 μm was used. In addition, as the through-hole 4 a formed in the insulating plate 4, a hole having a diameter of approximately 100 μm was formed.

In addition, instead of the cells A and B having different dielectrophoretic properties, particles D made of polystyrene beads having a diameter of approximately 10 μm were used.

As the alternating voltage, sine waveform voltages having amplitudes of 1, 1.2, and 1.5 V, a direct current component of 0 to 3 V, and frequencies of 10 kHz, 100 kHz, 500 kHz, 1 MHz, 5 MHz, and 10 MHz were used.

The results are shown in FIG. 6.

FIG. 6 shows the relationships among the frequency, direct current component, and amplitude of the alternating voltage, each relationship indicating conditions under which the particles D were made to stay still at a position away from the insulating plate 4 by a predetermined distance and corresponding to the through-hole 4 a formed in the insulating plate 4. That is, when the amplitudes of the alternating voltage were set to 1, 1.2, and 1.5 V, and the frequency was sequentially changed at the above individual amplitudes, the values of the direct current components at which the particles D were not attracted by either of the electrodes 3 a and 3 b were plotted.

At all amplitudes, since the value of a necessary direct current component was maximized in the vicinity of a frequency of 1 MHz, it was understood that a large electrophoretic force f2 was applied to the particles D. Hence, in the vicinity of a frequency of 1 MHz, it was understood that a large dielectrophoretic force f1 counteracting the large electrophoretic force f2 was exerted.

As a result, by adjusting the amplitude and the direct current component of the alternating voltage, the particles D are made to migrate toward the electrode 3 a by the electrophoretic force f2, and particles D to which the dielectrophoretic force f1 is applied continue to flow in the same way or are made to migrate in the opposite direction, so that separation can be performed.

In addition, in the above embodiment, by covering the surface of the electrode 3 a with the insulating plate 4, only the portion exposed through the through-hole 4 a was used as the electrode 3 a; however, an electrode (not shown) having a small area similar to that of the through-hole 4 a may be employed from the beginning.

Next, another Example using the cell separation device 1 shown in FIG. 1 will be described with reference to FIGS. 7 and 8.

As the device conditions, the distance between the electrodes 3 a and 3 b was set to approximately 1.2 mm, and as the insulating plate 4, a plate having a thickness of 0.2 mm was used. The width of the flow path 2 was set to 0.5 mm (the width of the flow path 2 a and that of the flow path 2 b were each set to 0.5 mm), and as the through-hole 4 a formed in the insulating plate 4, a hole having a diameter of approximately 0.2 mm was used.

As living cells and dead cells, 3-2H3 cells on the second day of culture were used, and as a bulk liquid, a mixture of an aqueous solution containing 8.5 percent by weight of sucrose and an aqueous solution containing 0.3 percent by weight of glucose was used.

FIG. 7 shows frequency properties of a living cell retention boundary when the amplitude of the alternating voltage was set to 80 V, and the frequency was changed to 1, 5, and 10 kHz. As shown in FIG. 7, it was found that the highest offset voltage was output at a frequency of 5 kHz, and in addition, the retention area was increased.

FIG. 8 shows retention boundaries and retention areas of living cells and dead cells when the frequency was set to 5 kHz, and the amplitude of the alternating voltage was changed to 60, 80, 100, and 120 V. When the conditions of the cell separation device 1 were set to obtain a separation possible area shown in FIG. 8, the dead cells were transmitted, and the living cells could be retained; hence, the living cells and the dead cells could be separated from each other.

In addition, still another Example using a cell separation device 111 shown in FIG. 9 will be described with reference to FIGS. 9 to 11.

As the device conditions, in the cell separation device 111 having a width of 12 mm, a length of 90 mm, and a height of 30 mm, the distance between the electrodes 3 a and 3 b was set to approximately 1.2 mm, and as the insulating plate 4, a plate having a thickness of 0.2 mm was used. The number of holes was set to approximately 1,000, and as the through-hole 4 a formed in the insulating plate 4, a hole having a diameter of approximately 0.2 mm was used. In the arrangement shown in FIG. 10, the distances between holes were set to 0.3 mm and 0.6 mm.

As shown in FIG. 9, a cell-suspension liquid was fed into the cell separation device 111 through a pump P and was transferred with a bulk liquid in a flow direction L. When the alternating voltage was applied by operation of an arbitrary waveform generator G, an electric field having an electric field strength gradient was formed between the electrodes 3 a and 3 b by operation of the insulating plate 4, and simultaneously, by the direct current component included in the alternating voltage, charges were unevenly distributed at one electrode side. For example, by this uneven distribution of charges, living cells received a dielectrophoretic force in the direction in which the electric field strength decreased, and to dead cells having no dielectrophoretic properties, the dielectrophoretic force was not applied but only the electrophoretic force was applied; hence the living cells and the dead cells could be separated at the retention side and the transmission side, respectively. Alternatively, when an electric field was formed so that, by this uneven distribution of charges, the living cells received a dielectrophoretic force in the direction in which the electric field strength increased, a dielectrophoretic force was not applied to the dead cells having no dielectrophoretic properties, but only the electrophoretic force was applied thereto; hence the living cells and the dead cells could be separated at the transmission side and the retention side, respectively.

FIG. 11 shows retention rates of living cells and dead cells when separation of a mixture containing living and dead cells was performed such that the frequency was set to 5 kHz, the amplitude of the alternating voltage was set to 100 V, and the offset voltage was output in the range of 0 to 1.0 V.

The flow rates of the cell-suspension liquid, bulk liquid, retention liquid, and transmission liquid were set to 0.3, 0.3, 0.2, and 0.4 ml/minute, respectively.

As the living cells and the dead cells, 3-2H3 cells on the second day after culture at a concentration of 5.0×10⁵ cells/ml were each used, and as the bulk liquid, a mixture of an aqueous solution containing 8.5 percent by weight of sucrose and an aqueous solution containing 0.3 percent by weight of glucose was used.

As shown in FIG. 11, with the cell separation device 111, since the offset voltage was output, the retention rate of the dead cells decreased, that is, the dead cells were transmitted; hence, the living cells, keeping a high retention rater could be separated from the dead cells having a decreased retention. 

1. A cell separation device comprising: a flow path through which a cell suspension flows, the cell suspension containing plural types of cells which have different dielectrophoretic properties; electrodes disposed to face each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path; an electric field gradient forming portion which generates an electric field strength gradient between the electrodes; and a power supply applying an alternating voltage having a direct current component across the electrodes.
 2. The cell separation device according to claim 1, wherein the electric field gradient forming portion is an insulating member which has at least one opening and which is disposed between the electrodes.
 3. The cell separation device according to claim 2, wherein the electrodes are parallel plate electrodes which form an electric field having an approximately uniform electric field strength.
 4. The cell separation device according to claim 1, wherein the electric field gradient forming portion is constructed by forming one of the electrodes is smaller than the other electrode.
 5. A cell separation method comprising the steps of: making a cell suspension containing plural types of cells having different dielectrophoretic properties flow in a flow path; and applying an alternating voltage, including a direct current component, across electrodes which face each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path to form an electric field having an electric field strength gradient between the electrodes. 